Influence of DOTA chelator position on biodistribution and targeting properties of 111In-labeled synthetic anti-HER2 affibody molecules

Anna Perols, Hadis Honarvar, Joanna Strand, Ramkumar Selvaraju, Anna Orlova, Amelie Eriksson Karlström, Vladimir Tolmachev

Research output: Contribution to journalArticlepeer-review

28 Citations (Scopus)


Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with 99mTc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule ZHER2:S1 at three different positions: DOTA-A1-ZHER2:S1 (N-terminus), DOTA-K58-ZHER2:S1 (C-terminus), and DOTA-K50-ZHER2:S1 (middle of helix 3). The affinity for HER2 differed slightly among the variants and the KD values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-ZHER2:S1, DOTA-K50-ZHER2:S1, and DOTA-K58-Z HER2:S1, respectively. ZHER2:S1-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-ZHER2:S1 (64 °C) and DOTA-K58-ZHER2:S1 (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with 111In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-ZHER2:S1and DOTA-K58-ZHER2:S1 showed the highest uptake. Overall, DOTA-K58-Z HER2:S1 provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

Original languageEnglish
Pages (from-to)1661-1670
Number of pages10
JournalBioconjugate Chemistry
Issue number8
Publication statusPublished - 15 Aug 2012
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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